The imaging of samples to provide identification and/or quantification of an analyte present within the sample is an important process in many branches of chemical and biological research. Analytes may be identified and quantified directly within a sample by measuring, for example, chemiluminescence or radioactivity; alternatively, the analyte may first require derivatisation with a detectable label, such as a fluorescent dye, prior to image analysis. Imaging devices consequently use a variety of detectors to identify and quantify such analytes, ranging from photodiodes, photomultiplier tubes (PMT) to charge coupled device (CCD) cameras. A variety of imaging devices are commercially available depending upon the desired modality (e.g. fluorescence, phosphorescence, storage phosphor imaging, chemiluminesence). These imaging devices are designed to image samples which have a variety of different formats such as microtitre or microwell plates, slides, membranes, tissue slices or sections, cuvettes, tubes, gels or cassettes.
US 2004/0071394, for example, describes an imaging system with a sample holder which is designed to hold multiple slides and has the dimensions of a microplate. GB2294320 concerns an imaging device used for studying bacterial colonies which are sealed within a gel cassette system. In this example, a laser beam is passed through the cassette to generate light scattering data which can be analysed to determine bacterial growth. Yet other imaging devices are disclosed in U.S. Pat. No. 6,159,425, together with support devices for sample containers which are to be imaged. The support devices comprise a holder which supports but does not enclose the sample container on a shelf structure.
The imaging of biological molecules such as nucleic acids and proteins is of particular importance in biological research. There is a continuing need to identify and quantify proteins and nucleic acids originating from a diverse range of organisms spanning simple bacteria to biologically complex mammals. Following the success of the human genome project in which the genetic sequence of the genome was determined, there is growing scientific interest in characterising the protein composition and protein function in man and other organisms.
The separation of biological molecules, such as proteins and nucleic acids, prior to or in parallel with their identification and quantification, can be achieved by a variety of techniques. Gel electrophoresis is a technique used to determine the size and subunit composition of proteins. An electric field is applied to a solution containing a protein and, because it is a charged molecule, the protein then migrates through a gel at a rate that depends on its net charge, size, and shape. The gel is then fluorescently stained to reveal the different protein components. The gel is imaged using a fluorescence imaging device to allow identification and quantification of the individual proteins. Two-dimensional gel electrophoresis is an advanced technique that can resolve with great accuracy more than 1000 different proteins and provide a virtual ‘map’ of all the proteins present in a solution.
Proteomic analysis utilising 2-D polyacrylamide gel electrophoresis (PAGE) protein separation is another technique which is widely used but is relatively low-throughput due to the time-consuming process of image analysis which is required to determine differential protein expression. A more efficient process, known as 2-D Difference Gel Electrophoresis, utilises fluorescent dyes to label proteins prior to 2-D PAGE, allowing multiple samples to be co-separated and visualised on one gel. Typically, three protein extracts (e.g. one control and two treated) are labelled with different fluorescent dyes (e.g. Cy2, Cy3 and/or Cy5) then combined and separated by 2-D PAGE. Images are then captured using the Cy2, Cy3 and/or Cy5 excitation wavelengths and the difference between the samples determined by image analysis software to allow identification and quantification of the protein components in each sample (Unlu et al., 1997, Electrophoresis, 18, 2071-77; Tonge et al., 2001, Proteomics, 1,377-396).
However, the scanning of gels which have been used for electrophoretic separation of biological molecules with conventional scanners is fraught with technical difficulties. Thus, for instance, the gels are typically laid out upon the glass window or platform of an imaging device such as a fluorescence scanner and the lid of the scanner closed prior to scanning. After the imaging process has been completed, which is generally some 30 minutes later, the gels must be carefully removed (if they are to be retained) and the window or platform washed thoroughly. As this cleaning process often involves the use of corrosive solvents and chemicals, considerable care must be taken to avoid user injury and/or damage to the imaging device. In order to ensure that the interior of the device is protected from these chemicals and solvents, the glass window or platform must be sealed to prevent access of the corrosive cleaning materials to vulnerable components within the body of the machine. Alternatively, the components of the imaging device must be constructed of materials which are known to tolerate the cleaning chemicals and solvents.
The use of corrosive chemicals and solvents within an instrument lab, as opposed to a fume hood, raises additional difficulties for the user. In some instances, the instrument may need to be housed within a fume hood which is wasteful of this expensive resource.
Another problem experienced with imaging systems of the prior art is that of ‘Newton's Rings’, an interference phenomenon caused by light passing through two sheets of glass. Newton's Rings are concentric rings which appear when two sheets of glass or clear plastic are held close together and almost in parallel to each other. The rings appear when the air gap between the sheets is of a certain size relative to the wavelength so that the light rays encounter interference.
Such phenomena generally occur when a sample, contained within a glass or a plastic retainer, is imaged directly on the glass platform of an imaging device, the light thus having to pass through several glass sheets. This can happen when, for example, a sample contained within a glass or plastic container/slide is placed on the glass platform of the imaging device. The problem may also arise if a gel is sandwiched between two glass sheets forming a cassette and this cassette is positioned on top of the glass platform of an imaging device. Cassettes of this nature are commonly used within the industry and are commercially available (e.g. The Gel Company, San Francisco, Calif., USA) to save operator time, improve gel uniformity and facilitate handling and storage. The net result is an inaccurate intensity reading due to the non-uniformity caused by the Newton's Rings formed by interaction between the glass platform of the imaging device and the glass sheet of the cassette.
Existing cassettes, of the type described above, can have limited use in terms of storage of a sample. Thus, for instance, gels may desiccate with time because the cassette is not sealed to the environment. Furthermore, contamination of the sample or gel can occur if efforts are not made to protect or seal it from its environment.
US 2005/0008541 describes a gel holder which can be used in a method for locating gel pieces using a scanner and removing the located pieces from the gel. The base plate of the holder is transparent to electromagnetic radiation in order to allow optical scanning or detection of concentration points in the gel. However, the gel holder does not comprise any locking device to seal the gel and thus prevent desiccation and contamination from the environment.
Caddies or structures for housing and holding multi-well platforms in a planar configuration to facilitate optical reading are disclosed in WO 1999/042608. The caddies can be adapted for robotic handling or optical reading and may be fitted with lids. However, no releasable locking devices are used to seal the caddies from the environment.
WO 2004/074818 describes a case for a microfluidic sample array which comprises a frame, top and bottom which, in operation, are sealed together to be tight to liquids. At least one of the top or bottom structures are light transmissive to facilitate optical reading. The case lacks any locking device which can be used to protect any sample enclosed within it, the component parts simply being sealed together, if required, after the sample is inserted within it.
The present invention seeks to address the aforementioned problems with the prior art and to provide solutions thereto, principally in the form of a method for imaging a sample, an imaging device and a frame for the device.